Histological tissue specimen preparation is a physical process that involves chemical solutions reacting with biological specimens. Typically specimens such as tissue samples from biopsies and autopsies require processing. The end result of such processing is a sample that has been preserved, and been infiltrated with paraffin. Once the tissue has been embedded in the paraffin, it is stable and may then be subsequently embedded and then sectioned on a microtome. This process has typically involved four different sub-procedures:
(a) Fixation
Fixation is a process by means of which cell proteins are stabilised, and the process is normally performed using chemical solutions. A good fixative is usually a fluid that will neither shrink nor swell the tissue, and more particularly will not dissolve its constituent parts, but will kill bacteria and render enzymes inactive. In addition, the solution should modify tissue constituents in such a way that they retain their form when subjected to treatment that would have damaged them in their initial state. The most commonly used chemical solution is formalin.
(b) Dehydration
Since the ultimate purpose of tissue specimen treatment is to infiltrate the tissue sample in paraffin, and since water and paraffin are not miscible, the sample must be dehydrated after the fixation step. This is usually achieved by subjecting the tissue sample to increasing concentrations of alcohols.
(c) Clearing
After dehydration, the tissue sample is still not capable of accepting paraffin since paraffin and alcohol are not miscible. A chemical solution, selected to be miscible with both alcohol and paraffin, is used to clear the alcohol from the sample. The chemical solution most commonly used is xylene. Unfortunately, xylene is considered to be toxic although most histological processing laboratories use xylene on a daily basis.
(d) Infiltration
The fourth and final step in the tissue sample treatment is infiltrating the sample, usually with paraffin wax. In this step the cleared tissue samples are placed into paraffin heated to a few degrees above its melting temperature. Several changes of paraffin may be required to remove the residual xylene so that the tissue is completely infiltrated with the molten paraffin.
The timing of the fluid change for all the fluids relates to the requirement to effectively displace the previous chemical from the tissue samples. Tissue samples can vary considerably in content and size, and therefore there may be a large variation in the time required to displace the fluid from one sample compared to the time taken to displace fluid from another. Further, some samples are sandwiched between biopsy pads that are porous and absorb significant quantities of fluid.
The first attempt at automation of the previously manual method of tissue processing involved placing solutions in a circular arrangement so that samples could be moved from container to container until they reached the last heated paraffin reservoir. The most well known instrument with this type of configuration used in the histology field was the Technicon. One of the major disadvantages of instruments of this type was that they allowed fumes to escape into the laboratory, thus exposing the laboratory workers to a hazardous environment. To overcome this problem, the next generation of tissue processing instruments included a centrally located closed chamber for the tissue samples. The solutions necessary for tissue processing were delivered into the closed chamber using suitable valving where the fluids are pumped in and out of the chamber in sequence. Normally the chamber would not be opened during the process.
As the chamber is closed, and only a single protocol can be run, the protocol must attempt to cater for the range of tissue samples that may be included in a single run. This can result in either over processing or under processing of some samples. Given the sealed nature of the retort, tissue samples may not easily be removed or added during a processing run.
Another problem is that some samples require urgent processing, while other samples are not urgent. In the known tissue sample preparation apparatus it has not been possible to stop a current sample run to process a sample required urgently, or to employ a protocol that allows an urgently required sample to be processed with other samples that require longer processing times. Thus, either the urgently required sample is run in isolation, or it is put with other samples, increasing the processing time.
Examples of known automated tissue processing machines will be found in the patent literature, and typical examples include U.S. Pat. No. 4,141,312 Louder, and U.S. Pat. No. 5,049,510 Repasi et al.
The prior art has therefore been unable to deal adequately with ensuring that a variety of samples can be processed safely and efficiently.
Some systems include heating of wax or tissue samples with microwaves, however microwave systems are difficult to automate, and preferentially heat the tissue sample rather than the reagents.
There is a need in the art for a tissue processor that provides automation to tissue processing.
There is also a need to reduce the use of hazardous chemicals employed in tissue processing.
There is a further need to clear infiltrating material of contaminants.